肿瘤微环境非编码RNA下调STING信号通路诱导T细胞功能耗竭的分子机制及其在鼻咽癌免疫治疗中的作用

The T-cell exhaustion is the main cause of tumor escape and the failure of tumor immunotherapy, and the imbalance of innate immunity and choric virus infection is linked to T-cell exhaustion in malignancies, however the main molecular mechanism of underway T-cell exhaustion in tumor microenvironments of nasopharyngeal carcinoma (NPC) is still largely unknown. We showed that the T-EXO and its carried miRNAs mediated the T-cell function impaired, Myeloid-derived suppressor cell (MDSC) expansion and the activity of STING signaling pathway which regulate the innate immune responses and was down-regulated in NPC in our previous study. Here, we supposed that T-EXO-carried non-coding RNAs regulate the T-cell exhaustion via down-regulating the STING signaling pathway in NPC. Firstly, we screen the different expression profile of T-EXO-carried miRNA/lncRNA in NPC by the microarray of non-coding RNA expression chip or RNA-sequencing array, then we analyze the regulations between the T-EXO-carried miRNAs/lncRNAs and their target genes in STING signaling pathway, and their effect on the MDSC differentiation and T-cell function through gene overexpression or knockout, FACS staining, immunoblotting or RNAchip et al. In addition, we will explore the immunotherapy strategy for NPC by small molecular drug targeting the potential target miRNA/lncRNA or target genes in STING signaling pathway by in vitro and in vivo analysis (xenograft mouse model). We expect to find out the biomarkers of prognosis or the potential targets for immunotherapy in NPC, and help to improve the clinical benefit of TIL immunotherapy of NPC patients.

T细胞功能耗竭是恶性肿瘤免疫逃逸和免疫治疗失败的主要原因，先天性免疫细胞失衡与慢性病毒感染是T细胞功能耗竭的主要诱因，但鼻咽癌微环境调控T细胞功能耗竭的分子机制至今尚不明确。我们前期发现鼻咽癌微环境肿瘤exosome（T-EXO）及其携带miRNA参与了T细胞功能失活的调控、髓源性抑制细胞（MDSC）数目的扩增，且与STING蛋白表达及其信号通路活性下调相关联。在本项目中，我们通过RNA表达芯片或测序技术、生物信息分析、基因编辑技术、免疫实验等方法筛查T-EXO富集的miRNA/lncRNA，探讨T-EXO富集miRNA/lncRNA靶向STING分子和信号通路的调控机制，及影响MDSC分化和参与T细胞功能耗竭的作用；最后通过动物实验探讨靶向非编码RNA或STING通路相关基因的小分子药物在鼻咽癌免疫治疗中的作用。我们预期发现鼻咽癌T细胞功能耗竭的调控机制、预后评估标志及潜在的免疫治疗靶点

当前研究认为STING通路是先天性免疫调控的主要信号通路，在肿瘤免疫中起了重要的作用。本项目以EBV感染的鼻咽癌为模型，探讨了肿瘤微环境中STING表达水平与肿瘤患者预后的相关性，调控STING信号通路的相关因素，以及肿瘤微环境中免疫细胞STING通路的激活对免疫细胞分化及功能的调控，最终阐明STING信号通路对肿瘤免疫逃逸和疾病转归的影响。我们主要发现了以下研究结果：1. STING通路在鼻咽癌组织表达下调，且STING的高表达与患者的良好预后相关；2.我们发现肿瘤微环境在缺氧的条件下上调miR-24，STING是miR-24的直接调控靶基因，miR-24可抑制STING的表达水平；3.鼻咽癌细胞STING通路的失活，可导通过p-STAT3通路导致炎性细胞因子如IL-1及IL-6的分泌增加，肿瘤微环境细胞因子的增加可促进髓系抑制细胞MDSC的分化，进而发生MDSC介导的T细胞免疫抑制，导致鼻咽癌的免疫逃逸；4.我们发现鼻咽癌高表达免疫检查点Tim-3的配体蛋白Galectin-9，Galectin-9可通过外泌体分泌至细胞外，外源性Galectin-9可抑制CD33+细胞的STING的表达，CD33+细胞内源性STING通路的失活，也可通过细胞因子分泌的改变，导致CD33+细胞向MDSC分化，导致鼻咽癌免疫逃逸，5.我们发现与外周PBMC相比肿瘤浸润T细胞的STING通路被激活，T细胞内源性STING通路的激活可诱导Treg的分化（宫颈癌模型），导致宫颈癌肿瘤Treg的扩增。6.我们发现包括鼻咽癌在内的多种肿瘤细胞系上调lncRNA-NEAT1，NEAT1可抑制STING分子的表达，因而抑制肿瘤细胞的铁死亡，导致肿瘤细胞的免疫耐受。总的来说，本研究发现了肿瘤微环境STING分子及信号通路在浸润免疫细胞的分化及功能中起了重要的作用，参与肿瘤的免疫监控及免疫逃逸的调控；但STING的功能具有细胞特异性，且与肿瘤的起源有相关性，因而后续，STING激动剂及靶向STING在肿瘤免疫治疗中的作用还需进行深入的机制研究。